The life cycle of retroviruses involves an obligatory stage in which the virus genetic material is inserted into the genome of a host cell by transposition-like events. This step is essential because the inserted viral nucleic acid, the provirus, is replicated through the host cell machinery.
Because retroviruses have genomes of diploid single-stranded RNA (ssRNA), those must be replicated through a double-stranded DNA intermediate prior to their insertion. The initial conversion of the viral RNA molecule into a double-stranded DNA (dsDNA) molecule is performed by a reverse-transcriptase. The dsDNA is then led to the nucleus, where one or more copies are integrated to the genome by an integrase to further be replicated by the host cellular machinery. The reverse transcriptase and the integrase required for the conversion of ssRNA into dsDNA and for the integration into the host genome are carried within the viral particle during host cell infection. The proviral DNA is finally transcribed using the host machinery into multiple RNA copies. These RNA molecules will further be translated into virus peptides or integrated into virus particles which will be released from the cell into the extracellular milieu.
Retrovirus RNA molecules usually comprise 6 typical regions leading to the expression of multiple proteins by processing reactions These typical retrovirus region include the gag, pol and env gene sequences associated to a psi (ψ) signal and flanked by 5′ and 3′ long terminal repeats (LTR) sequences. The gag gene leads to the expression of the protein components of the nucleoprotein core of the virus, while the pol gene products are involved in the synthesis of nucleic acid and recombination. The env gene codes for the envelope components of the retrovirus particle. 5′ and 3′ LTR sequences of those genes ensure the correct transcription of the virus RNA into DNA and subsequent integration of the virus genome into the chromosomal DNA of the cell. Finally, the psi signal refers to the retroviral packaging signal that controls the efficient packaging of the RNA into the virus particle, and thus to gene transfer.
Because of their ability to form proviruses, retroviruses appeared as adequate tools to modify the genome of particular cells for uses such as gene therapy. Gene therapy using retroviral vectors is generally performed by adding an exogenous nucleic acid sequence to the retroviral RNA vector, packaging this vector into a virus particle and infecting a target host cell. The target cell will then incorporate the exogenous gene as being a part of a provirus.
For safety reasons, retroviral vectors must be replication incompetent, since the target cell would suffer from a retroviral infection. The use of vector systems now allows the production of recombinant retroviruses that are unable to replicate by themselves. Those systems make use of virus that comprise the exogenous gene of interest, flanked only by the minimal sequences required for retrotranscription into DNA, insertion into the host cell genome and proper expression of this gene. The RNA molecule carried by the non-replicative retroviral particle is devoid of gag, pol and env genes and therefore, the target cell does not produce the nucleoprotein core nor the envelope essential to the replication of the virus.
To infect target cells, encapsidation of the exogenous RNA molecule however remains a necessary step. Therefore, a complementation system must be provided in packaging cells. The virus assembly is performed in packaging cells infected with helper virus or now more generally with viruses transiently or stably transfected with constructs comprising psi-negative gag, pol and env genes.
Transfection of a psi-positive construct comprising the exogenous gene into such infected or transfected packaging cells leads to the encapsidation of the RNA molecule into a virus. Next, the packaging cells release the retroviral vector particles, or virus, into the supernatant. As gag-pol and env genes are not carried by the virus particle, those genes cannot be transferred from the packaging cells to the target cells.
Packaging cells are frequently designed to express retroviral vectors that are derived from the Moloney murine leukemia virus (MLV). In fact, MLV-derived vectors are the most commonly used vectors in clinical trials for gene therapy. Although these replication-defective recombinant retroviruses can be produced by transient cotransfection of an expression vector comprising the exogenous gene and packaging plasmids coding for gag-pol and env viral proteins, the absence of toxicity of MLV proteins has made possible the generation of stable retrovirus-producer cell lines which are convenient for use in large-scale vector production. To improve the infectious properties of recombinant retroviruses, env gene products of more infectious viruses can be used. This process, known as pseudotyping, is commonly used to modify the virus tropism to make it more infectious and/or more specific to a specific cell type. For example, retroviral vectors pseudotyped with the feline leukemia RD114 env glycoprotein have been shown to be very promising for gene therapy, particularly since they show resistance to complement inactivation and are efficient to transfer genes into human lymphocytes and hematopoietic stem cells.
One major safety concerned with stable packaging cell lines is to ensure that expression vectors generated from these packaging cells are not contaminated with replication-competent retroviruses (RCRs). RCRs result from the recombination between the expression vector and the packaging plasmids. It has also been shown that non human primates can develop lymphomas after being grafted with genetically modified hematopoietic stem cells contaminated with RCR. To prevent such deleterious recombinations, the latest generations of packaging cells use an expression vector and a packaging plasmid that have reduced overlapping homologies in retroviral sequences, reducing the risk of generating replication competent viruses.
Although the latest generations of packaging cell lines are effective in protecting from RCR contamination, the scale up for clinical uses is limitative because of the adherence of those cells. Indeed, since the growing environment of cells is limited to the surface of a recipient, the production of large volumes of retroviruses is cumbersome and can be quite expensive. In addition, most of the cells actually used for packaging require the use of animal serum for their growth. This represents another drawback for the existing packaging cell lines because of the biohazard contamination risks. Finally, murine cells used in the production of MLV particles can produce a characteristic α-galactosyl epitope at the surface of the virus. This epitope can be recognized by the immune system of the host organism, causing an antibody-mediated inactivation of the recombinant retrovirus particles.
Recently, a human embryonic kidney (HEK) cell line, the 293SF cell line, has been developed (U.S. Pat. No. 6,210,922) and is a good candidate to bypass the above-mentioned drawbacks generally associated with packaging cells. First, these cells are human cells and therefore do not produce the carbohydrate structure α-galactosyl epitope found at the surface of the virus produced from murine cells. In addition, the genomic DNA from these cells does not hybridized with MLV specific probes at low or high stringency, therefore precluding the generation of RCR by recombination with endogenous retrovirus, as found with murine packaging cell lines. Stable packaging cells have already been derived from HEK 293 cells and it seems that they have the property to produce recombinant retroviruses with relatively high titers. Finally, 293SF cells grown in suspension in a serum-free media are already available and are used for the large scale production of proteins and adenoviral vectors.
Considering the state of the prior art, it would be desirable to be provided with a packaging cell line that grows in suspension with serum-free medium and is capable of expressing high titers of recombinant retrovirus particles precluding the generation of RCR.